1. Field of the Invention
The present invention relates to a process for producing 5-methyluridine, a material useful as a synthetic intermediate for medications such as azidothymidine, a commercially available AIDS medication, and d4T (2',3'-dihydroxy-2',3'-dideoxythymidine), a medication which is undergoing clinical testing for treatment of AIDS.
2. Discussion of the Background
Currently, 5-Methyluridine is chemically synthesized and purified via crystallization from an organic solvent such as methanol, ethanol or the like (see for example, J. Am. Chem. Soc., vol. 78, p. 2117 (1956); Japanese Laid-Open Patent Application (Kokai) No. 63-63668; Helvetica Chimica Acta, p. 2179 (1980) and Synthesis, p. 259 (1982)).
5-methyluridine can also be produced by reacting a nucleoside or ribose-1-phosphoric acid with 5-methyluracil in the presence of a microorganism (Japanese Laid-Open Patent Application (Kokai) No. 2-23882 (hereinafter JP 2-23882)). However, this patent does not describe how to purify 5-methyluridine and is therefore insufficient for the production of 5-methyluridine as the main product.
It is desirable to form large crystals of 5-methyluridine via crystallization so as to increase the separability of the crystals, reduce a size of the device required for the separation and improve the separation from the impurities. However, there is no teaching in JP 2-23882 to describe the same.
Ordinarily, crystal growth is influenced by the extent of supersaturation, by large particle diameter and by impurities. The composition of the impurities varies greatly depending on the system to be crystallized. Controlling the composition of the impurities is important in making crystallization possible. The specific control needed in any given system varies.
Thus, with respect to the formation of 5-methyluridine using a microorganism, although JP 2-23882 describes how to produce 5-methyluridine, it fails to describe how to purify 5-methyluridine from the reaction solution.
When 5-methyluridine was crystallized from the reaction solution of JP 2-23882 by concentration after slight pretreatment, it was found that 5-methyluridine crystals having a particle diameter of just from 20 to 30 micrometers (.mu.m) were obtained, which decreased the separation rate and increased the size of the separator. It was further found that crystals having such a particle diameter cannot be suitably separated from impurities on the basis of a difference in the particle diameter of crystals which will be described later.
Generally speaking, in order to obtain crystals having a uniform particle diameter industrially, a method is adopted in which the fluid to be crystallized is allowed to flow by itself from a lower portion, crystals having a large diameter are divided into a lower portion, crystals having a small diameter are divided into an upper portion, and the fine crystals in the upper portion are dissolved outside the system and recycled. However, processes in which product crystals are separated from impurity crystals based on the difference in a precipitation rate (sedimentation velocity) as in the present invention are few. This is because there is no system having such a particle diameter distribution; a system in which as impurity crystals are included in product crystals, and even if the product crystals can be separated from the impurity crystals, the purity would not increased; and even if crystals have a particle diameter or qualities that enable classification, nobody has yet arrived at a classification system.
When 5-methyluridine is produced from nucleic acid ingredients as starting materials via an enzyme reaction using a microorganism, the reaction product contains unreacted nucleic acid ingredients and nucleic acids formed as by-products. When 5-methyluracil and guanosine are used as starting materials to produce 5-methyluridine, thymine, guanine, guanosine and 2-amino-7-.beta.-ribofuranosyl-7H-purine-6(1H) one (pseudoguanosine) mainly exist as impurities in the reaction solution. Of these, for example, thymine is similar to 5-methyluridine with respect to a pattern of a change in a solubility relative to temperature and pH. Therefore, it is difficult to remove thymine via crystallization. Further, thymine is similar to 5-methyluridine with respect to the ionization pattern as well. Therefore, it is difficult to remove thymine via treatment with an ion-exchange resin.
These nucleic-acid-type impurities are peculiar to a process wherein the nucleic acid ingredients are employed as starting materials and the reaction is conducted using a microorganism. Thus, ordinary knowledge cannot be utilized, nor does known literature describe a method of separating these ingredients. It is desirable to find a method for purifying 5-methyluridine from a reaction conducted using a microorganism.